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The effect of transcription factors on TAD boundaries in AML and upon myeloid differentiation - The HOXA locus

van Midden, Wouter (2019) The effect of transcription factors on TAD boundaries in AML and upon myeloid differentiation - The HOXA locus. Research Project 1, Biomedical Sciences.

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Acute Myeloid Leukemia (AML) is a heterogeneous malignancy, characterized by impaired differentiation towards the myeloid lineage and an increased self-renewal ability. One of the most frequently mutated genes in AML is NPM1 (NPM1cyt), which is associated with aberrant HOXA expression, and in particular increased HOXA9 expression. ChIP-seq on primary AML patients samples in combination with HiC data showed the presence of a topologically associated domain (TAD) between the HOXA10 and HOXA11 genes on the HOXA locus. The majority of AML patients associated with an intact TAD boundary carried the NPM1mut. This TAD boundary was also present in healthy CD34+ cells, and was lost upon myeloid differentiation. Expression of HOXA9 was typically associated with an intact TAD boundary as seen in normal CD34+ cells and in leukemic blasts of NPM1cyt AML patients. NPM1wt AML patients have often lost the TAD boundary, as well as HOXA9 expression. Analyzing the TCGA dataset showed that myeloid transcription factors (TFs) IRX1, CEBPa, and PU.1 possibly had a potential effect to remove TAD boundaries. Therefore, our hypothesis is that reintroduction or overexpression of TF activity in cells that have an intact TAD boundary will result in loss of the TAD boundary, repression of HOXA gene expression, and myeloid commitment. TAD boundaries are associated with CTCF binding sites and the cohesin complex, which consists out of four core-units: RAD21, STAG2, SMC1A, and SMC3. Therefore, we hypothesized that accelerated differentiation of CD34+ can be initiated by knockdown of the cohesin genes or the CTCF gene to an impaired function of the TAD boundary. Overexpression of CEBPa and PU.1 did not result in altered genetic expression on the HOXA locus, and did not alter the histone marks present on these loci. Furthermore, it did not induce differentiation of OCI AML3 cells. IRX1 overexpression in HEK 293T cells showed a decrease in HOXA9 expression levels, downregulated H3K4me3 levels, and a decreased CTCF and RAD21 binding at the CTCF binding site. These results suggests that the TAD boundary was (at least partly) removed by IRX1 overexpression. Unfortunately, IRX1 overexpression in CD34+ cells did not result in altered differentiation towards any lineage. KD of CTCF did not result in altered RAD21 binding at the TAD boundary, but resulted in highly upregulated HOXA genes in HEK 293T cells. Altogether, these results shows that IRX1 could be a potential candidate to alter TAD boundaries in hematopoietic malignancies and upon differentiation.

Item Type: Thesis (Research Project 1)
Supervisor name: Schuringa, J.J.
Degree programme: Biomedical Sciences
Thesis type: Research Project 1
Language: English
Date Deposited: 01 Nov 2019
Last Modified: 04 Nov 2019 10:04

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